Primary antibody against

To detect expression of SMURF2 in the excised tumor sections, immunofluorescence staining was conducted according to standard procedures. To obtain tumor sections, all tissues were embedded in Optimal Cutting Technique (OCT) compound (Leica, Bensheim, Germany), and immunofluorescence staining was conducted using primary antibody against SMURF2 (Santa Cruz; 1 : 100), followed by Alexa Fluor 549‐conjugated goat anti‐rabbit IgG (Thermo Fisher; 1 : 500).
To access the induction of apoptosis in vivo, TUNEL staining was conducted in the fixed tumor sections using an in situ cell death detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. To visualize the nucleus, the tumor sections were mounted with DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA) and evaluated using a ZEISS LSM 700 confocal microscope (Oberkochen, Germany).

Cells were grown directly on chamber slides before immunofluorescence. After washing with phosphate-buffered saline (PBS), cells were fixed in in 4% paraformaldehyde for 20 min at room temperature. After fixation, cells were washed three times in PBS for 5 min. Subsequently, cells were incubated with primary antibody against IRF3 (anti-rabbit; Santa Cruz Biotechnology) and NFKB (anti-mouse; Santa Cruz Biotechnology) at dilution 1:100, overnight at 4 °C. Next day, cells were washed three times in PBS for 5 min and incubated with secondary antibodies: TRITC (anti-rabbit) and FITC (anti-mouse) at dilution 1:200 for 1 h at room temperature. The slides were mounted with medium containing DAPI (4,6-diamidino-2-phenylindole) to visualize nuclei. The fluorescent images were obtained using a Zeiss Axio Imager Z1 Microscope with Apotome 2 system (Zeiss, Milan, Italy).